Cell surface receptors respond to a plethora of extracellular stimuli under several physiological or pathological conditions. Key components of the network, connect and diversify into multiple signal transduction pathways, further amplified by enzymes, cofactors and transcription factors. o2h scientists can quantitatively access drug-target engagement by measuring a variety of intra-cellular signaling events

Some examples include cAMP, Ca²⁺, inositol phosphates, phosphoproteins, transcriptional regulation and sub-cellular protein translocation. These events can be sensitively captured using kit-based or bespoke assay designs utilizing different detection platforms like FLIPR, high-content imaging, western blotting, real time qPCR and plate readers.

As a case example, we have highlighted below our experience in GPCR biology wherein there might be an opportunity to screen compounds that are agonists, partial agonists or antagonists for neuropsychiatric disorders that target serotonin receptors.

Serotonin 2A receptor (5-HT2A) plays a key role in mood regulation, cognition, and perception and is activated by the neurotransmitter serotonin. A prototypical psychedelics active Gq/11-mediated activation of PLC, leading to the formation of inositol phosphates, diacylglycerol, followed by Ca2+ release, and β-arrestin2 making their respective roles still unclear.

At o2h we have developed and screened a series of 5-HT2A-small molecules with varying potencies towards Gq and β-arrestin-biased signaling thererby shedding light on the dynamics associated with the 5-HT2A receptor. These serve as invaluable tools in elucidating the specific intracellular responses providing comprehensive understanding for drug discovery and pharmacological research.

Target 5HT2A Receptor

Hallucinogens like LSD, psilocybin, and substituted N-benzyl phenylalkylamines although widely used recreationally, are considered therapeutic for many neuropsychiatric disorders in the treatment of depression and anxiety. Their mechanism of action, both therapeutic and hallucinogenic are not completely understood, although the modulation of 5-HT2A receptor (5-HT2AR) is the key.

5-HT2AR is a G-protein coupled receptor (GPCR) that canonically activates Gq signalling initiating a Phospholipase C (PLC)-mediated breakdown of the membrane phospholipid, phosphatidylinositol bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 mobilizes intracellular Ca2+ stores in the endoplasmic reticulum that activates calcium/calmodulin dependent kinase II (CaMKII); whilst DAG activates protein kinase C (PKC), which phosphorylates extracellular signal-regulated kinase (ERK) and the transcription factor cyclic AMP response element-binding protein (CREB), amongst others to regulate gene expression. The 5-HT2AR driven Gq signalling pathway is postulated to mediate behavioural responses to hallucinogens, as evidenced by the absence of the classical hallucinogen-induced head-twitch response in 5-HT2AR knockout mice. Further, biochemical and mouse mutant studies support a heterocomplex of the Gq-coupled 5-HT2AR and the Gi-coupled metabotropic glutamate receptor (mGlu2R) in the frontal cortex in mediating the cellular and behavioural responses induced by hallucinogens.

While the 5-HT2AR canonically activates heterotrimeric Gq stimulating intracellular release of Ca2+, the receptor also interacts with β-arrestin proteins controlling the efficacy and duration of its signalling. The coupling mechanisms can vary from one brain region to another, differentially activating particular brain regions that may be associated with specific neurochemical, physiological, and behavioural responses. Understanding the aforementioned biased mechanisms and outcome in the context of drug discovery for the 5HT receptor subtypes, provides a strategy to achieve superior therapeutic outcomes in treating neurological disorders.

Figure 1: (A) Chemiluminescent detection of β-Arrestin recruitment in U2OS PathHunter cells in response to 5-HT (serotonin). The increase in serotonin concentration results in elevated luminescence, demonstrating a dose-dependent β-arrestin recruitment to the 5-HT2A receptor. The analysis was performed using non-linear regression, agonist vs. response variable slope (four parameters).
(B) U2OS PathHunter cells stimulated with indicated concentration of compounds and assayed for β-Arrestin recruitment.
Compounds exhibit differential, dose-dependent increase in luminescence – indicating their ability to recruit β-Arrestin to 5-HT2A receptor

Figure 2: (A, B) Stably expressing 5HT2A cells stimulated with an Increasing concentration of serotonin, and measuring intracellular Ca2+ (by FLIPR) and IP1 levels (by HTRF kit) respectively
(C) Compounds classified as agonists exhibiting different potencies and efficacies

Figure 3: (A) Cells treated with increasing concentration of the antagonist ketanserin exhibiting reduced IP1 generation upon stimulation with EC80 concentration of 5-HT (serotonin)
(B, C) Screening of compounds for their antagonistic activity by measuring intracellular levels of IP1 and Ca2+ respectively

Figure 4: NanoBiT assay for the interaction of 5HT2a receptor with beta arrestin 2 (barr2). (A) Schematic representation of the NanoBiT assay. The 5HT2a receptor is C-terminally tagged with LgBiT whilst the barr2 is N-terminally tagged with SmBiT. The addition of native ligand 5HT (serotonin) causes an interaction of the two proteins and structural complementation of luciferase to generate a luminescent signal.
(B) Luminescence readings from 2 hr kinetic read on cells transfected with 5HT2aR-LgBiT and SmBiT-barr2. Solid blue lines represent samples with 1uM 5HT added at 0 mins, whilst dashed blue lines represent control samples with only buffer added. The total DNA amount transfected (5HT2aR and barr2 plasmids) was reduced as a 2-fold dilution and presented as a blue heatmap showing decreasing DNA amount.
(C) Relative area under the curve (AUC) of graphs in B. Note whilst the total DNA decreases, the fold change upon addition of 5HT remains consistent. Negative control is cells transfected with 5HT2aR-LgBiT and HaloTag-SmBiT which show no interaction upon 5HT addition.

o2h discovery assay setup and cell lines

• Our o2h scientists can support in identifying compounds with differential signaling activities, downstream of the 5-HT receptor, stimulated via Gαq and beta-arrestin pathways
• Stable cell lines overexpressing the receptor of interest e.g. 5HT2A/2B/2C and for different species – human, rat and mouse can be developed in appropriate cellular background for screening. These cells can measure IP₁ and Ca²⁺ as a stable down stream signal utilizing FLIPR to address activation of Gαq pathway.
• PathHunter cell lines from Eurofins/Discoverx can be utilized for the screening of 5HT2A agonists, antagonists and partial agonists in bespoke assays with a transferable license agreement with DiscoverX
• These cells overexpress human 5-HT2A receptor and the β-arrestin2. These proteins are engineered to reconstitute β-galactosidase activity upon recruitment of β-arrestin2 to 5-HT2A receptor
• We have explored the PathHunter cell line for IP one measurements as well using HTRF-based IP₁ kit from CisBio. A significant and reliable fold window is achieved for both IP1 and beta Arrestin with the PathHunter cell line, making it convenient to study biased signalling
• o2h can also support in exploratory studies using BRET-based protein-protein interactions or complementation assays (an area that is currently gaining interest). The study will involve cloning into appropriate vector systems (e.g. LgBiT-SmBiT), based on your biology of interest and customize them to your needs

To know more about our biology services offering or to request our brochure, please reach out to us at discovery@o2h.com.